Relevance of JAK2V617F positivity to hematological diseases - survey of samples from a clinical genetics laboratory

<p>Abstract</p> <p>Background</p> <p>JAK2V617F is found in the majority of patients with Ph- myeloproliferative neoplasms (MPNs) and has become a valuable marker for diagnosis of MPNs. However, it has also been found in many other hematological diseases, and some studies even detected the presence of JAK2V617F in normal blood samples. This casts doubt on the primary role of JAK2V617F in the pathogenesis of MPNs and its diagnostic value.</p> <p>Methods</p> <p>In the present study, we analyzed JAK2V617F positivity with 232 normal blood samples and 2663 patient blood, bone marrow, and amniotic fluid specimens obtained from a clinical genetics laboratory by using a simple DNA extraction method and a sensitive nested allele-specific PCR strategy.</p> <p>Results</p> <p>We found JAK2V617F present in the majority (78%) of MPN patients and in a small fraction (1.8-8.7%) of patients with other specific hematological diseases but not at all in normal healthy donors or patients with non-hematological diseases. We also revealed associations of JAK2V617F with novel as well as known chromosomal abnormalities.</p> <p>Conclusions</p> <p>Our study suggests that JAK2V617F positivity is associated with specific hematological malignancies and is an excellent diagnostic marker for MPNs. The data also indicate that the nested allele-specific PCR method provides clinically relevant information and should be conducted for all cases suspected of having MPNs as well as for other related diseases.</p

Generation of a new congenic mouse strain with enhanced chymase expression in mast cells.

Mast cells are effector cells best known for their roles in IgE-associated allergy, but they also play a protective role in defense against pathogens. These cells express high levels of proteases including chymase, tryptase and carboxypeptidase. In the present study, we identified a congenic strain of C57BL/6 mice expressing an extraordinarily high level of chymases Mcp-2 and Mcp-4 in mast cells. The overexpression was associated with variant Mcp-2 and Mcp-4 genes originated from DBA/2 mice that also expressed high levels of the two enzymes. Real time PCR analysis revealed that Mcp-2 and Mcp-4 were selectively overexpressed as tryptases, Cpa3 and several other chymases were kept at normal levels. Reporter gene assays demonstrated that single-nucleotide polymorphisms (SNPs) in the promoter region of Mcp-2 gene may be partly responsible for the increased gene transcription. Our study provides a new model system to study the function of mast cell chymases. The data also suggest that expression of chymases differs considerably in different strains of mice and the increased chymase activity may be responsible for some unique phenotypes observed in DBA/2 mice

Association of Mcp-2 and Mcp-4 overexpressions in BMMCs with gene variants.

<p><b>A.</b> Mice were genotyped for the Mcp-2 gene variant by allele-specific PCR and the Mcp-4 gene variant by restriction fragment length polymorphism (RFLP) with NdeI. BMMCs derived from these mice were analyzed for Mcp-2 and Mcp-4 protein expressions by Commassie blue staining and western blotting. <b>B.</b> BMMCs from B6 and B6-cma mice were analyzed for chymase activity. In at least 45 mice analyzed, there is a perfect correlation of Mcp-2 and Mcp-4 gene variants with overexpression of Mcp-2 and Mcp-4 and increased chymase activity.</p

Expression of mast cell proteases in mast cells derived from B6, B6-cma, and DBA/2 mice.

<p>Expression of indicated mast cell proteases together with house-keeping gene GAPDH (glyceraldehyde 3-phosphate dehydrogenase) was analyzed by real time PCR using specific PCR primers shown in the top panel. Data represent relative gene expression levels calculated based on threshold cycles and standard curves obtained with serial dilutions of purified PCR products. Error bars denote standard deviation (n≥3). *p<0.0001 in comparison with B6 control mice.</p

Chymase activity in mast cells from B6, B6-cma, and DBA/2 mice.

<p>Mast cells were derived from bone marrow (BM) and peritoneal cavity (PC) of B6, B6-cma, and DBA/2 mice. Cells were either extracted for assays of total chymase activity (A) or treated with antigen to induce degranulation for determination of secreted chymase activity (B). Specific activity was calculated in reference to total proteins in cell pellets. Error bars denote standard deviation (n≥3). *p<0.0001 in comparison with B6 control mice.</p

Reporter gene assays.

<p><b>A.</b> Schematic diagram of report gene constructs. SNPs are indicated, and black blocks represent CANNTG motifs (E-boxes). <b>B.</b> Report gene constructs were transfected into mast cells derived from bone marrow of B6 mice. <b>C.</b> Report gene constructs together with pcDNA3 plain vector or pcDNA3-Mitf-A were transfected into NIH3T3 cells. Relative report gene expression is represented by firefly luciferase activity normalized against that of renilla luciferase. Error bars denote standard deviation (n≥3).</p

Comparison of Mcp-2 and Mcp-4 protein levels in mast cells from B6, B6-cma, and DBA/2 mice.

<p>Mast cells were derived from bone marrow (BM) and peritoneal cavity (PC) of B6, B6-cma, and DBA/2 mice. <b>A.</b> Cell extracts were resolved on 12.5% SDS gels followed by Coomassie blue staining (top panel) or western blotting with indicated antibodies. <b>B.</b> Cells were subjected to Wright-Giemsa staining (top panel) or immunofluorescent staining with indicated antibodies.</p

Evaluation and characterization of volatile air toxics indoors in a heavy polluted city of northwestern China in wintertime

Hazardous volatile organic compounds (VOCs) and carbonyls were evaluated in typical dwellings in Xi'an in northwestern China in wintertime. High indoor concentrations were observed for formaldehyde, acetone, naphthalene, methylene chloride and acetaldehyde, associated with characteristic pollution sources. In comparison, many of the target VOCs were higher in Chinese dwellings than those in other countries, suggesting the significances of indoor pollutions in China. Source apportionment with receptor model shows that furniture and building materials (44.5%), paints and adhesives (11.9%), household products (17.3%), smoking (14.5%), and cooking (9.8%) are the major contributors to the indoor VOCs and carbonyls. The health risk assessment shows that the cancer risks for formaldehyde (5.73 x 10(-5)), 1,3-butadiene (2.07 x 10(-5)) and 1,2-dichloroethane (1.44 x 10(-5)) were much higher than the acceptable level of 1 x 10(-6) recommended by International Register for Certified Auditors (IRCA). The hazard quotient (HQ) of target VOCs were far less than the threshold (HQ = 1). Moreover, the practical efficiency of household air purifier in removal of the VOCs and carbonyls was examined first time in dwellings in northern China. The results prove that most of the indoor organic pollutants and their cancer risk to humans can be efficiently reduced, particularly for formaldehyde and 1,3-butadiene. The findings of the study offer useful preliminary and updated information on current indoor air toxics levels, dominant pollution sources and their potential health risks to residents in northwest China